As stated by R. Scott Mclvor (Molecular Therapy, May 2011, 19(5):822-3, Therapeutic Delivery of mRNA: The Medium Is the Message), it seems as though every advance in gene transfer and expression needs to somehow provide a solution to the problem of genotoxicity. This relies on developing an alternative to gene transfer methods involving a DNA intermediate, which necessarily present a potential risk of insertional mutagenesis, no matter whether they are intended to be integrative or not. For instance WO 2005/116225 describes retroviral vectors producing DNA, the aim of these vectors being to avoid DNA integration into the host genome. However, due to the production of DNA, the risk of detrimental insertional mutagenesis persists due for example to host cell dependent recombination mechanisms. The present invention relates to a transient vector involving no DNA intermediate.
Messenger RNA (mRNA) has several advantages over DNA for gene transfer and expression, including the lack of any requirement for nuclear localization or transcription and the nearly negligible possibility of genomic integration of the delivered sequence.
However, the development of mRNA as therapeutic faces the same challenge as any nucleic acid: delivery. It is therefore likely that substantial improvements will be required in the efficiency of mRNA delivery and translation into protein product to reach a level that is of more general therapeutic utility.
The present invention now advantageously addresses these drawbacks and provides a new solution for the use of mRNA as a source of gene product usable in vitro, ex vivo or in vivo for both therapeutic or non-therapeutic (for example, research and transgenesis) applications. The present invention more widely answers the long-felt need for safe, efficient and transient gene transfer tools as further developed below.
Transiently transferring a nucleic acid or a protein, also herein respectively identified as “recombinant nucleic acid” or “transgene” and “recombinant protein”, into a target cell is a major issue in the biotechnological field, in particular in a therapeutic or experimental context.
In the context of therapy, such a transient expression may be mandatory, for instance, for safety reasons, i.e., in order to avoid deleterious effects of a sustained expression of the recombinant protein in the subject exposed to this therapy or to prevent permanent integration of the recombinant nucleic acid into the host genome by preventing the generation of DNA intermediate forms of the recombinant nucleic acid.
In the context of non-therapeutic uses, the transient expression may be required, for example, in order to avoid deleterious effects such as positional effects or genomic toxicity. For example, in the context of transgenesis, the transient expression of a DNA-modifying enzyme may be advantageous in the zygote or early in the development of the organism to specifically modify a target locus. In this context, avoiding DNA intermediate forms encoding the DNA-modifying enzyme in the organism would advantageously prevent potential integration of the genome encoding the DNA-modifying enzyme, consequently preventing constitutive expression of said DNA modifying enzyme in said organism.
One may further wish to limit in vitro, ex vivo or in vivo the expression of a recombinant peptide, for example when such expression is only transiently required at a particular moment of a biological process.
Among the technical options currently available to allow the transient delivery of a nucleic acid or a peptide, the skilled person may select either a non-viral or a viral delivery method. The non-viral delivery method may be a direct peptide delivery method or a direct nucleotide delivery method. The viral delivery method may imply the use of replicative RNA viruses, such as RNA (±) viruses and RNA (−) viruses, or transpackaging of fusion proteins in non-replicative retroviral vectors.
Non-Viral Delivery Methods
Direct delivery of protein is chosen in specific contexts, such as for the delivery of a ligand of a cell membrane receptor in vitro or ex vivo. However, in most cases, if the action of the delivered factor is to be intracellular, the efficiency of direct delivery of protein in the culture media may be limiting and other methods should be considered. In addition, direct protein delivery does not allow for targeting a specific cell type, which is a particular issue for in vivo applications.
According to another method used in the art, nucleotide sequences (DNA and/or RNA) can be directly delivered to cells by mean of current transfection protocols based on chemical or physical methods, for in vitro, ex vivo or in vivo uses.
The cationic polymers (e.g., diethylaminoethyl (DEAE)-dextran, poly(l-lysine), dendrimers, polyethylenimine (PEI)) and the lipid vectors (e.g., liposomes or lipoplexes such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) are examples of chemical compounds used to transfer nucleic acids.
Physical methods can also be considered for the transient delivery of nucleic acids into a target cell, such as electroporation. For DNA transfer, the strength of the electric shock required is high enough to allow the material to reach the cell nucleus. Such a shock may however be responsible for cell damage.
One can also consider delivering nucleic acid molecules by direct micro-injection into the target cells. This method is mainly applied for transgenesis purposes. It can also be used for gene transfer into poorly permissive cells. Micro-injection is limited to in vitro applications and it is a tedious method, highly time-consuming (cell by cell), and has not been automated so far for most cells.
These non-viral methods can be of interest under certain circumstances such as transient gene transfer into cell lines in vitro or, for example, in the muscle in vivo. However, they are in most cases of limited efficiency, do not allow targeting of a specific cell type and are not easily implemented since they require large amounts of highly purified recombinant nucleic acid and some of the recombinant nucleic acid based compositions, complexed with lipid compounds for instance, are particularly unstable and cannot be stored. In addition, in the case of plasmid DNA, bacterial sequences are retained that can have deleterious effects.
Viral Delivery Methods of RNA
Viruses are nucleoprotein particles transferring their nucleic information into a target cell and hijacking it for their own replication. For years, they have been modified to be used for gene transfer purposes in vitro, ex vivo and in vivo. “Viral vectors”, also herein identified as “virus-derived vectors” or “vector particles”, are mostly deprived of virulence factors and non-replicative. They further allow the expression of a sequence of interest: the transgene. They often overcome the lack of efficiency of non-viral gene transfer methods, taking advantage of the strategies viruses have developed over their evolution.
Viruses can be classified in several families, depending on the nature of their nucleic acid content and how it is processed during the viral cycle: RNA viruses contain RNA molecules ((+), (−) or double-stranded), DNA viruses contain DNA molecules (single- or double-stranded) and reverse transcribing viruses have their genome as RNA or DNA depending on the step of the replication cycle.
Among RNA viruses, RNA (+) viruses can be used for transient transfer of RNA because they are never present in the cell as a DNA molecule, i.e., they do not have DNA intermediates, thus precluding in theory any persistence of the viral information and subsequent mutagenic risk. Their genome is processed in the cell cytoplasm like a messenger RNA and is translated by cellular ribosomes for the production of viral proteins. One can thus modify them to carry only the information of interest. Viral vectors have been developed from several RNA (+) viruses including alphavirus (e.g., Sindbis, Semliki forest virus, Venezuelan equine encephalitis), picornavirus (e.g., poliovirus), or flavivirus (e.g., Kunjin virus).
Among those kinds of RNA (+) virus derived vectors, alphaviruses are preferred because of their ability to transduce a large quantity of exogenous proteins into cells of a large range of species. However they exhibit cytopathic effects precluding their use for most therapeutic purposes.
The RNA (−) viruses group comprises most of the main human pathogens, including flu, rabies and measles viruses. The RNA (−) genome is not infectious on its own but has to be associated with a RNA-dependent RNA-polymerase, which will generate the (+) strand.
While several viruses of this family have been used to develop RNA vectors, including measles, the most used one is the Sendai virus (SeV), a paramyxoviridae. The Sendai virus is not genotoxic and can replicate in a large range of mammalian tissues. It is the causative agent of respiratory infections in mice, guinea pigs, hamsters, rats and in rare cases in pigs, but is not pathogenic for humans. Sendai-derived vectors are used in a few therapeutic strategies, for instance in cystic fibrosis mouse models.
Nevertheless, one of the major drawbacks of these vectors derived from RNA (+) and RNA (−) viruses is the replication of the RNA genome and the formation of non-transmissible virus-like particles (NTVLP) into the host cell, leading to toxicity and cell death in many cases.
Viral Delivery Methods of Protein (Trans-Packaging of Fusion Proteins with HIV-1 Derived Particles)
An approach to transiently bring a protein into a target cell consists of the trans-packaging of a protein of interest in vector particles derived from HIV-1. This trans-packaging can be achieved by fusion with naturally encapsidated proteins.
Among the viral proteins investigated as trans-packaging partner is VPR (Viral Protein R), which has various functions in the HIV-1 cycle and pathogenicity. The fusion of a heterologous protein of interest to VPR allows one to encapsidate this protein. Such a strategy has for instance been tested for trans-complementation assays to study the functions of integrase and reverse transcriptase during the retroviral cycle. It can also be used to vectorise therapeutic proteins.
The main drawback of this VPR trans-packaging lies in the primary functions of VPR itself regarding cell cycle arrest and its pro-apoptotic and cytotoxic effects, all properties which originally drove the removal of VPR from the improved generation of HIV-1 vectors and which are retained in the fusion proteins. Another disadvantage is the relatively poor efficiency of trans-packaging, depending on the nature of the fusion protein.
Integrase is another viral protein that has been recently used, with mitigated successes, for trans-packaging of heterologous proteins in HIV-derived particles, to visualize cell/particle interactions (fusion with reporter genes), to develop targeted integration vectors (fusions with specific DNA-binding domains such as LexA or ZFN), and to vectorise proteins of interest (fusion with p53).
Based on fusion with either VPR or integrase (IN) or any other viral protein, the efficiency of trans-packaging strategy is limited by important factors: the low number of proteins that can be delivered and the functionality of the fused protein. Indeed, although the number of encapsidated VPR proteins is high in regard to the particle, the packaging efficiency of a fusion protein is expected to be lower, due to the limited size of the particle; this is also true for IN fusion proteins. This low delivery efficiency can, in particular cases, preclude the use of this method. In addition, a protein which is fused to VPR or IN may lose its function. In consequence, fusion is to be designed with caution in a way not to alter the protein of interest.
The present invention now offers a solution to the problems of the art and provides new tools and vectors for transiently expressing a transgene in vitro, ex vivo or in vivo with optimal safety.